The goals of this proposal are the following: 1) Determine protein- protein interactions between Polypyrimidine Tract Binding protein (PTB), PTB associated Splicing Factor (PSF) and other splicing factors in vivo using the yeast two hybrid system. 2) In vitro and in vivo domain analysis involved in protein-protein interactions between PTB and PSF. 3) Determine the optimal RNA binding sequences for PTB and PSF and the complex between the two using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assays. PTB and PSF potential interactions may be involved in splice site selection, particularly in the alpha- tropomyosin (alpha-TM) gene in smooth muscle cells (SMCs). The transition from the normal contractile state to a proliferative state is marked by a change in exon selection. In normal smooth muscle cells, exon 2 is retained while in proliferating cells exon 3 is retained. Initiation of SMC proliferation within the arterial wall is accompanied by a change in exon selection and characteristic of early development of atherosclerosis. The studies described in this proposal will elucidate how the interactions between PTB and PSF may potentially regulate alternative splicing of alpha-TM in SMCs.